Figure 3. Binding evaluation of the anti-maytansinoid sdAbs to DM4.

(A) Affinity characterization of the derivative anti-maytansinoid single-domain antibodies (sdAb) to free DM4. Binding activity of the sdAbs against free DM4 was evaluated via competitive enzyme linked immunosorbent assay (ELISA). The concentration of each anti-DM4 sdAb was held constant, and free DM4 at concentrations ranging from 0.1 nM to 1 μM were added. Solutions were incubated for 30 minutes before being added to an ELISA plate coated with DM4-biotin-avidin. The fraction bound (%A/A₀) of the sdAbs to immobilized DM4 decreased with increasing concentrations of free DM4. Points represent the mean of samples in triplicate with standard deviations depicted by the error bars. All clones isolated from the mutated library have moderate to high affinity to free DM4, with IC50 values ranging from 10 nM to 56 nM. The calculated binding IC50s are reported in Table S3. (B) Anti-maytansinoid sdAbs potently inhibit DM4 cytotoxicity. SK-BR-3 cells were incubated with DM4 (100 pM – 1000 nM) with or without co-incubation with 10 μM of purified anti-maytansinoid sdAb over a 24 h exposure period, following with viability assay to assess cell cytotoxicity. The results were normalized to cells treated with vehicle control and reported as % cell viability. The data was fitted for cytotoxic IC50 values with Hill slope in GraphPad Prism software. Each data point represents the mean of triplicate samples with standard deviations shown with error bars. The IC50 of DM4 applied to SK-BR-3 cells was between 0.3 to 0.4 nM. In the presence of 10 μM of purified anti-DM4 sdAbs, DM4 cytotoxicity was reduced up to 250-fold. Calculated IC50s are reported in Table S3.